Cholesteatomas of the middle ear and mastoid develop as a complication of otitis media. These epidermal structures often become infected with a mixture of aerobic and anaerobic bacteria, the most common of which is Ps. aeruginosa. Bacteria form biofilms within cholesteatomas resulting in chronic infection. These infected cholesteatomas are aggressive and cause increased osteolysis. We have identified strains of otopathogenic Ps. aeruginosa (OPPA) that have increased adherence to keratinocytes and increased biofilm production. We will further characterize these isolates by examining the expression of quorum sensing genes and alginate gene expression and production. We propose to study the pathogenesis and virulence of these chronic infections. With regard to pathogenesis, we will study the adherence of these organisms to keratinocytes using randomly directed mini-Tn5 transposon mutagenesis of adherent OPPA isolates. The mutagenesis screen will be enriched for non-adherent mutant bacteria and will subsequently be sequenced to identify novel adhesin genes. With regard to virulence, preliminary studies show that our OPPA strains produce osteoclastogenesis by both an LPS-dependent and LPS-independent mechanism. LPS-dependent studies will be done with LPS sensitive murine osteoclast precursors. Expression of signals associated with osteoclastogenesis (e.g. RANKL, TNFa) will be determined to understand the mechanism of Ps. aeruginosa LPS mediated osteoclastogenesis. To study LPS-independent osteoclastogenesis, we will use LPS-insensitive osteoclast precursors derived from mice deficient in toll-like receptor 4 to determine which portions of the osteoclast development pathway are induced.